Electric birefringence studies demonstrate differences between filaments of dephosphorylated and phosphorylated myosin II that are interpreted as indicating that filamentous dephosphorylated myosin II is much less flexible around the hinge region. Dephosphorylated monomers and filaments are more rapidly cleaved within the globular head than phosphorylated monomers and filaments. This provides additional evidence for a conformational difference between the dephosphorylated and phosphorylated molecules, even as monomers. This structural difference in monomers is reflected in an in vitro motility assay: phosphorylated monomers are inactive and dephosphorylated monomers fully active. The three myosin I isoforms are differentially localized in the cell: myosin IA is mostly in the actin-rich cortex, myosin IB and IC in the plasma membrane and large vacuole membranes, and only myosin IC in the contractile vacuole membrane. Myosin I heavy chain kinase binds to purified plasma membranes where its autophosphorylation and activity are activated, membrane-bound kinase phosphorylates membrane-bound myosin I and the phosphorylated myosin I has enhanced actin-activated ATPase activity. In vitro, Ca-calmodulin inhibits phospholipid-stimulated autophosphorylation of the kinase and, hence, its activity by competing for the phospholipid binding site. This provides a possible mechanism for Ca regulation of actomyosin I activity in vivo.